Fluorescent calcium sensors are widely used to image neural activity. Using structure-based mutagenesis and neuron-based screening, we developed a family of ultrasensitive protein calcium sensors (GCaMP6) that outperformed other sensors in cultured neurons and in zebrafish, flies and mice in vivo. In layer 2/3 pyramidal neurons of the mouse visual cortex, GCaMP6 reliably detected single action potentials in neuronal somata and orientation-tuned synaptic calcium transients in individual dendritic spines. The orientation tuning of structurally persistent spines was largely stable over timescales of weeks. Orientation tuning averaged across spine populations predicted the tuning of their parent cell. Although the somata of GABAergic neurons showed little orientation tuning, their dendrites included highly tuned dendritic segments (5–40-µm long). GCaMP6 sensors thus provide new windows into the organization and dynamics of neural circuits over multiple spatial and temporal scales.
At a glance
- Depolarization and calcium entry in squid giant axons. J. Physiol. (Lond.) 218, 709–755 (1971) , &
- Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans. Neuron 26, 583–594 (2000) et al.
- Spatially resolved calcium dynamics of mammalian Purkinje cells in cerebellar slice. Science 242, 773–777 (1988) , , &
- The life cycle of Ca2+ ions in dendritic spines. Neuron 33, 439–452 (2002) , &
- Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex. Nature 433, 597–603 (2005) , , , &
- Multiple dynamic representations in the motor cortex during sensorimotor learning. Nature 484, 473–478 (2012) et al.
- In vivo dendritic calcium dynamics in neocortical pyramidal neurons. Nature 385, 161–165 (1997) , , &
- Activity in motor-sensory projections reveals distributed coding in somatosensation. Nature 489, 299–303 (2012) et al.
- Stereotyped odor-evoked activity in the mushroom body of Drosophila revealed by green fluorescent protein-based Ca2+ imaging. J. Neurosci. 24, 6507–6514 (2004) et al.
- Dendritic organization of sensory input to cortical neurons in vivo. Nature 464, 1307–1312 (2010) , , &
- Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nature Methods 6, 875–881 (2009) et al.
- A Cre-dependent GCaMP3 reporter mouse for neuronal imaging in vivo. J. Neurosci. 32, 3131–3141 (2012) et al.
- Characterization and subcellular targeting of GCaMP-type genetically-encoded calcium indicators. PLoS One 3, e1796 (2008) , , , &
- A genetically encoded reporter of synaptic activity in vivo. Nature Methods 6, 883–889 (2009) , , &
- Heterogeneity in synaptic transmission along a Drosophila larval motor axon. Nature Neurosci. 8, 1188–1196 (2005) et al.
- Optimization of a GCaMP calcium indicator for neural activity imaging. J. Neurosci. 32, 13819–13840 (2012) et al.
- A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein. Nature Biotechnol. 19, 137–141 (2001) , &
- Mank, M. et al. A genetically encoded calcium indicator for chronic in vivo two-photon imaging. Nature Methods (2008). 5, 805–811
- Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887 (1997) et al.
- Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins. Proc. Natl Acad. Sci. USA 101, 10554–10559 (2004) , , , &
- Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs. Chem. Biol. 13, 521–530 (2006) et al.
- Spontaneous network activity visualized by ultrasensitive Ca2+ indicators, yellow Cameleon-Nano. Nature Methods 7, 729–732 (2010) et al.
- Monitoring neural activity and [Ca2+] with genetically encoded Ca2+ indicators. J. Neurosci. 24, 9572–9579 (2004) , &
- In vivo performance of genetically encoded indicators of neural activity in flies. J. Neurosci. 25, 4766–4778 (2005) et al.
- Circular permutation and receptor insertion within green fluorescent proteins. Proc. Natl Acad. Sci. USA 96, 11241–11246 (1999) , &
- Molecular and structural basis of target recognition by calmodulin. Annu. Rev. Biophys. Biomol. Struct. 24, 85–116 (1995) &
- Crystal structures of the GCaMP calcium sensor reveal the mechanism of fluorescence signal change and aid rational design. J. Biol. Chem. 284, 6455–6464 (2009) et al.
- Target recognition by calmodulin: dissecting the kinetics and affinity of interaction using short peptide sequences. Protein Sci. 5, 1215–1228 (1996) , &
- Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nature Biotechnol. 22, 1567–1572 (2004) et al.
- Highly selective receptive fields in mouse visual cortex. J. Neurosci. 28, 7520–7536 (2008) &
- Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex. Nature 420, 788–794 (2002) et al.
- Synaptic calcium transients in single spines indicate that NMDA receptors are not saturated. Nature 399, 151–155 (1999) , &
- Functional mapping of single spines in cortical neurons in vivo. Nature 475, 501–505 (2011) , , , &
- A quantitative map of the circuit of cat primary visual cortex. J. Neurosci. 24, 8441–8453 (2004) , &
- Functional specificity of local synaptic connections in neocortical networks. Nature 473, 87–91 (2011) et al.
- The mechanism of orientation selectivity in primary visual cortex without a functional map. J. Neurosci. 32, 4049–4064 (2012) &
- Broad inhibition sharpens orientation selectivity by expanding input dynamic range in mouse simple cells. Neuron 71, 542–554 (2011) et al.
- Orientation selectivity of synaptic input to neurons in mouse and cat primary visual cortex. J. Neurosci. 31, 12339–12350 (2011) , , , &
- NMDA receptor subunit-dependent [Ca2+] signaling in individual hippocampal dendritic spines. J. Neurosci. 25, 6037–6046 (2005) , &
- GABAergic neurons are less selective to stimulus orientation than excitatory neurons in layer II/III of visual cortex, as revealed by in vivo functional Ca2+ imaging in transgenic mice. J. Neurosci. 27, 2145–2149 (2007) , , , &
- Broadly tuned response properties of diverse inhibitory neuron subtypes in mouse visual cortex. Neuron 67, 858–871 (2010) , , &
- Network anatomy and in vivo physiology of visual cortical neurons. Nature 471, 177–182 (2011) et al.
- Differential connectivity and response dynamics of excitatory and inhibitory neurons in visual cortex. Nature Neurosci. 14, 1045–1052 (2011) et al.
- Ca2+ imaging of mouse neocortical interneurone dendrites: Ia-type K+ channels control action potential backpropagation. J. Physiol. (Lond.) 551, 49–65 (2003) , &
- Total number and ratio of excitatory and inhibitory synapses converging onto single interneurons of different types in the CA1 area of the rat hippocampus. J. Neurosci. 19, 10082–10097 (1999) , , &
- Calcium microdomains in aspiny dendrites. Neuron 40, 807–821 (2003) , , &
- Roller Coaster Scanning reveals spontaneous triggering of dendritic spikes in CA1 interneurons. Proc. Natl Acad. Sci. USA 108, 2148–2153 (2011) et al.
- An expanded palette of genetically encoded Ca2+ indicators. Science 333, 1888–1891 (2011) et al.
- Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics. Front. Mol. Neurosci. 6, 2 (2013) et al.
- Deep tissue multiphoton microscopy using longer wavelength excitation. Opt. Express 17, 13354–13364 (2009) et al.
- Structural basis for calcium sensing by GCaMP2. Structure 16, 1817–1827 (2008) , , &
- Efficient gene transfer into rhesus repopulating hematopoietic stem cells using a simian immunodeficiency virus-based lentiviral vector system. Blood 103, 4062–4069 (2004) et al.
- Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 6, 343–345 (2009) et al.
- Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA 9, 493–501 (2003) et al.
- Optical recording of action potentials in mammalian neurons using a microbial rhodopsin. Nature Methods 9, 90–95 (2012) , , , &
- Ephus: multipurpose data acquisition software for neuroscience experiments. Front. Neural Circuits 4, 100 (2010) et al.
- Nonlinear dendritic integration of sensory and motor input during an active sensing task. Nature 492, 247–251 (2012) et al.
- A developmental switch in the response of DRG neurons to ETS transcription factor signaling. PLoS Biol. 3, e159 (2005) et al.
- The psychophysics toolbox. Spat. Vis. 10, 433–436 (1997)
- The VideoToolbox software for visual psychophysics: transforming numbers into movies. Spat. Vis. 10, 437–442 (1997)
- ScanImage: Flexible software for operating laser-scanning microscopes. Biomed. Eng. Online 2, 13 (2003) , &
- A pyramid approach to subpixel registration based on intensity. IEEE Trans. Image Process. 7, 27–41 (1998) , &
- Dichotomy of functional organization in the mouse auditory cortex. Nature Neurosci. 13, 361–368 (2010) , &
- Simple Neurite Tracer: open source software for reconstruction, visualization and analysis of neuronal processes. Bioinformatics 27, 2453–2454 (2011) , &
- Video 1: Simultaneous recording of spikes and GCaMP6s fluorescence signal of a L2/3 neuron in the visual cortex in vivo (12,818 KB, Download)
- This video shows simultaneous recording of spikes and GCaMP6s fluorescence signal of a L2/3 neuron in the visual cortex in vivo. The soundtrack of the video encodes the recorded spikes.
- Video 2: Calcium imaging of dendritic spines of GCaMP6s-expressing V1 neurons in response to drifting grating stimulation (19,513 KB, Download)
- This video shows 320 s of continuous calcium imaging of dendritic spines of GCaMP6s-expressing V1 neurons in response to drifting grating stimulation (2 s duration, 0.05 cycles per degree, 1 Hz temporal frequency). The direction of grating movement is indicated by the arrow in the upper right.
- Supplementary Information (7.1 MB)